One child in two hundred is born with limb deformities. Our understanding of the teleology of these defects, whether induced by teratogenes, caused by stochastic dysfunctions of the developmental program, or the result of congenital mutations, will require a deep understanding of the normal process of limb development. Many of the genes that are suspected of mediating the formation of limbs are being identified. Prominent among these genes are Hox, Fgfs, Wnts and Tbx genes. We are studying selected members of each of these gene families to determine their roles during limb development. Gene targeting in mouse embryo-derived stem cells provides the means for generating mice with designed modification in these genes, which include loss-of-function mutations, conditional mutations, gain-of-function mutations, lineage analysis of cells expressing these genes and gene swaps. The goal of this grant application is to use gene targeting to undertake a systematic genetic analysis of these genes and thereby establish their roles, individually and in combination with each other, during limb development. With respect to the Hox genes, emphasis will be directed at determining their potential role in limb induction and patterning of the principal elements of the limb. With respect to Fgfs, our emphasis will be placed on the roles of Fgf4 and Fgf8 in maintaining the proximodistal outgrowth of the limb. Finally, we wish to determine which Wnts are selectively used in the formation of the fore- and hindlimb buds and further testing the hypothesis of whether Tbx5/4 are used to specify the identity of fore- and hindlimbs. Through epistasis and molecular genetic analysis, it is hoped that we will gain further insight on how members of these separate families of genes function together as a network to determine the overall structure of the limb.